Immobilization of enzymes to synthetic polymer is of particular interest because of their application in clinical laboratories, biosensors, membrane bioreactors and diagnostics [Krysteva, M. A., Shopova, B. L., Yotova, I. Y., and Karasavova, M. I. (1991) Biotechnol Appl Btochem. 13, 106-111; Pandey, P. C., Kayastha, A. M., and Pandey, V. (1992). Appl. Biochem. Biotechnol. 33, 139-144]. There are different methods for immobilization of biomolecules on to a polymer surface e.g. entrapment, encapsulation, adsorption, covalent binding etc. Covalent immobilization is often necessary for binding molecules that do not adsorb, adsorb very weakly, or adsorb with improper orientation and conformation to polymer surfaces. This may result in better biomolecule activity, reduced nonspecific adsorption, and greater stability [Bangs, L. B., and Meza, M. B. (1995) Microspheres, part 2: Ligand attachment and test formulation. MD & DI's IVD Technol. 20-26; Larsson, P. H., Johansson, S. G. O., Hult, A., and Gothe, S. (1987) J Immunol Meth. 98, 129-135; Rasmussen, S.R., Larsen, M. R., and Rasmussen, S. E. (1991) Anal Biochem. 198, 138-142; Chevrier, D. Rasmussen, S. R., and Guesdon, J. L. (1993) Molec Cell Probes. 7, 187-197]. There are a number of ways to modify solid supports for the covalent immobilization of biomolecules. Aleixo et al. (1985) introduced an active functional group to an inert polystyrene surface by nitrating the aromatic ring of the polystyrene followed by the reduction of the nitro group to amino group; the amino polystyrene was further activated by a chemical reaction such as diazotization for covalent immobilization of protein.
Immobilization of biomolecules onto an inert polypropylene (PP) or polyethylene (PE) surface is of special interest because of their inability to bind through adsorption (unlike polystyrene) and their insolubility in most of the organic solvents. However, immobilization of biomolecules onto PP and PE surfaces is restricted due to the absence of any active functional group on them for chemical bonding. Therefore, it is necessary to activate these surfaces for immobilization of biomolecules. Activation of PP and PE surfaces has been reported to occur through radiation graft polymerization or gaseous plasma technique [de Queiroz et al., (1997) J. Biomater. Sci. Polym. 8, 667-681; Kaetsu, I. et al. (1980) J. Biomed. Mater. Res. 14, 199-210; Sipehia, R. et al. (1988) J Biomed. Mater. Res. 22, 417-422; Sipehia, R. (1998-99) Biomater. Artif. Cells Artif. Organ 16, 955-966.; Hayat, U. et al. (1992) Biomaterials 13, 801-806 and Wang, C. C. et al. (1993) J. Biomater. Sci. Polym. 4, 357-367]. For example, functional groups were introduced on the PE surface by copolymerization of ethylene with acrylic acid or acrylamide using gamma rays from a 60Co source for immobilization of albumin [de Queiroz et al., (1997) J. Biomater. Sci. Polym. 8, 667-681; Wang, C. C. et al. (1993) J. Biomater. Sci. Polym. 4, 357-367 and Sano, S. et al. ?( ) 20 993) Biomaterials 14, 817-822]. PP and PE surfaces can also be activated by introducing hydroxyl or amino groups by a plasma technique employing oxygen [Sipehia, R. (1998-99) Biomater. Artif. Cells Artif. Organ 16, 955-966] anhydrous ammonia [Sipehia, R. et al. (1988) J Biomed. Mater. Res. 22, 417-422; Hayat, U. et al. (1992) Biomaterials 13, 801-806] or decylamine hydrochloride [Terlingen, J. G. et al. (1993) J. Biomater. Sci. Polym. 4, 165-181]. The immobilization of enzymes such as glucose oxidase, peroxidase, and protein A and antibody on surfaces having an amino group has been achieved through a glutaraldehyde coupling method [Sipehia, R. et al. (1988) J Biomed. Mater. Res. 22, 417-422; Hayat, U. et al. (1992) Biomaterials 13, 801-806].
Guire had activated aminoalkyl matrix thermochemically with 1-fluoro-2-nitro-4-azidobenzene (FNAB) in 16-64 hours followed by attachment of the enzyme through the photochemical reaction in 2-16 hours [Patric, G. (1976) Method. Enzymol. 44, 280-288; U.S. Pat. No. 3,959,078]. Similarly, U.S. Pat. No. 4,973,493 describes a method where hydroxyl-bearing support was activated thermochemically with 1-fluoro-2-nitro-4-azidobenzene. Major drawbacks of the above procedures are that both the steps (activation and immobilization) are time consuming and cumbersome. Moreover, method of Guire gives the limiting choice of the matrix i.e. matrix must have an amino or similar nucleophillic group. Other major drawbacks are that the reactive nitrene, formed under UV exposure binds randomly with almost all the ingredients present there including biomolecule, matrix or solvent resulting in undesirable reactions which may lead to inefficient immobilization.
The photochemical method permits the covalent attachment of active functional group onto inert solid surface under gentle reaction conditions [Amos, R. A. Anderson, A. B., and Clapper, D. L. (1995) In Encyclopedic Handbook of Biomaterials and Bioengineering. Part A: New York, Marcel Dekker, 895-926]. This method is normally based on a compound having at least two functional groups of which one is essentially a photoactivable group. There are different methods for activation of inert surfaces through a photolinker. But in most of the methods, either the photoactivable compound is expensive or difficult to prepare or the procedure is cumbersome and time consuming.
In U.S. Pat. No. 4,973,493 describe the method for the modification of a polymer surface by attaching biocompatible agents like growth factor, fibronectin, collagen etc by using N-oxy succinimide esters of 4-azido-2-nitrophenyl epsilon amino caproic acid, 4-azido-2-nitrophenyl amino undecanoic acid and benzoylbenzoic acid. However, these modified surfaces are used for specific purpose like cell attachment.
U.S. Pat. No. 5,427,779 describes a method for the modification of an inert surface by a two-ring heterocyclic photolinker such as psoralenes in a photochemical reaction (2 hour irradiation at 350 nm). Biotinylated psoralenes were also used.
Disadvantages of these methods are i) posralenes are difficult to synthesize, ii) expensive, iii) require long irradiation time for bonding with the support and iv) during activation with biotinylated psoralenes, activated polymer is not generally applicable.
U.S. Pat. No. 6,033,784 and PCT no. WO 96/31557 discloses a method for photochemical modification using quinones. Using primary amino containing quinone derivative amino group was introduced onto polystyrene surface by UV irradiation followed by washing and drying for 50 minutes at 60° C. Similarly, acid group was introduced onto the polystyrene surface. These groups can not easily bind with the protein without addition of one or more activating reagent. More over preparation of such plates and photolinker are time consuming and cumbersome. These activated surfaces were also not suitable for protein binding or ELISA.
None of the above methods gives an activated surface, which is stable in light and capable of forming a covalent bond with the biomolecule more preferably, protein molecule. Since all of these methods are tedious and time consuming, there is a need for a convenient and direct technique allowing introduction of active functional groups onto inert surfaces which immobilize biomolecule by directly reacting with the nucleophilic group of the biomolecule.